Spinach is a dioecious plant where a male plant with male flowers, a female plant with female flowers and an intermediate plant with both male and female flowers are present. It is estimated that the mode of inheritance has a similar mechanism to XY type generally seen in animals and the sex determining genes are located on chromosome 1 as multiple alleles (Ellis and Janick 1960, Am. J. Botany 47, 210-214; Iizuka and Janick 1962, Genetics 47, 1225-1241; Sugiyama and Suto, Bull. Nat. Inst. Agr. Sci. (Japan) Series D, No. 11, 211-329 (1964)).
For such dioecious plants, it is important to identify the sex of individuals obtained by screening in a breeding process rapidly in order to improve the efficiency of breeding operations. However, the male and female of spinach cannot be definitely judged until the morphological differences between the male and female clearly appear after bolting, because the difference in karyotype between male and female spinach is not so clear as in the animal to make the discrimination of the sex chromosome difficult.
Conventional methods for early identifying the sex of plants include those using DNA markers such as RAPD (Random Amplified Polymorphic DNA) marker, SCAR (Sequence Characterized Amplified Regions) marker, etc. The RAPD method which is a technique based on the PCR method, was developed by Williams (Nucleic Acids Res 18, 6531-6535 (1990)), and for its relatively easy procedures, this method became rapidly widespread for plants as the main object. However, unexpected experimental errors can occur depending on DNA purity, PCR unit, etc., since the annealing temperature (35 to 42.degree. C. ) in the RAPD method is set lower than in the conventional PCR method. On the other hand, the SCAR method was developed by Paran and Michelmore (Theor Appl Genet 85:985-993 (1993)) in which a region with less experimental errors is amplified using PCR primers synthesized on the basis of a nucleotide sequence of a RAPD marker.
Although these DNA markers are extremely useful means for identifying the sex of plants, there are very few cases ever reported including White Campion (Melandrium album Garcke) and Pistachio(Pist acia vera L.) (Mulcahy et al., Sex Plant Reprod, 5, 86-88 (1992); and Hormaza et al., Theor Appl Genet, 89, 9-19 (1994)), and there is none of such reports on spinach.